PrP 106-126

Cell experiment:

The toxicity of PrP peptides is investigated on monolayers of PBEC. 100 000 PBEC/cm2 are seeded on 96 [for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] or 24-well plates [for lactate dehydrogenase (LDH)] pre-coated with rat tail collagen (27 μg/mL). The cell monolayers are treated with either PrP 106-126 wt or scr peptides. The PrP peptides are dissolved in double distilled water and then diluted in assay medium or in 5% newborn calf serum in Dulbecco-modified Earl's medium without phenol red. Treatment duration with the PrP peptides varies from 24 h to 48 h and the peptide concentration is set to 100 μM at which it exerts optimal effects as shown in a dose-dependent experiment. This concentration is also chosen as similar concentrations of PrP 106-126 found to exert in-vitro effects on astrocytes, neurons, microglia and leukocytes. The MTT assay, which measures mitochondrial metabolism, is based on the conversion of the water-soluble MTT dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble purple formazan. This formazan is then solubilized in pure dimethylsulfoxide, and its concentration determined by optical density (550/630 nm). The LDH cell death assay is done using the CytoTox 96® Assay kit. The released LDH in culture supernatants is measured with a 30-min coupled enzymatic assay which results in the conversion of a tetrazolium salt, 2-p-iodophenyl-3-p-nitro-phenyl-5-phenyl tetrazolium chloride (INT) into a red formazan product. The LDH levels are measured at the end of each treatment period. Thus, 50 μL of the medium is removed at the end of the treatment with PrP peptides and transferred to a new 96-well plate to which the other compounds are added. The plates are analyzed using an Elisa reader with the 490 nm filter.

References:

[1]. Yang W, et al. PRAS40 alleviates neurotoxic prion peptide-induced apoptosis via mTOR-AKT signaling. CNS Neurosci Ther. 2017 May;23(5):416-427.
[2]. Cooper I, et al. Interactions of the prion peptide (PrP 106-126) with brain capillary endothelial cells: coordinated cell killing and remodeling of intercellular junctions. J Neurochem. 2011 Feb;116(4):467-75.